Cas9 protein/gRNA transfection reagent
GenomONE TM-GE EX
|Product name||Cat. #||Specification||Frequency of use
|Freeze-dried HVJ-E||Reagent F,G||Buffer|
|GenomONE-GE EX||GG001EX||1 vial||each 1 vial||1 vial||325|
|GG004EX||4 vials||each 1 vial||1 vial||1,300|
|GG016EX||16 vials||each 4 vials||2 vials||5,200|
GenomONE-GE EX enables efficient Cas9 protein/gRNA transfection in a variety of difficult-to-transfect cells.
In case of plasmid DNA transfection for Cas9 and gRNA expression, please use GenomONE-GX EX .
*This product is sold for research purpose only. It may not be used for treatment or other clinical purposes or for intra- and extracorporeal diagnosis in humans or animals.
Features of GenomONE-GE EX
- Applicable for CRISPR-Cas9-mediated knock-out and knock-in
- Applicable for difficult-to-transfect cells (e.g., Mouse primary T cell, U-937, Jurkat)
- Simple operability (completion of transfection within 10 min)
Efficient delivery of Cas9 protein and gRNA into mouse primary T cells
T cells isolated from splenocytes were transfected with Cas9 protein sgRNA (2’OMe and PS modifications) using HVJ-E (GenomONE-GE EX) and product C or T (competitors). Genome editing efficiency was evaluated using T7 endonuclease I mismatch cleavage assay two days after transfection.
Induction of cell death by delivery of Cas9 protein and lethal gRNA targeting thousands of locations in the genome
Cas9 protein and Edit-R Lethal control gRNA (Horizon Discovery) were delivered into mouse primary T cells and Jurkat cells using GenomONE-GE EX and product C or T (competitors). Cell viability was measured by wst-8 assay (A450) two days after transfection. The GenomONE-GE EX efficiently delivered Cas9 protein and gRNA into mouse primary T cells and Jurkat cells, which are known as difficult-to-transfect cells.
Highly efficient ssODN-mediated homology directed repair (HDR)
Cas9 protein, sgRNA and ssODN (donor DNA) were transfected into HeLa, A549, HL-60 and U-937 cells using GenomONE-GE EX, respectively. ssODN (66base) was designed to carry the recognition site of BamHI restriction enzyme. After transfection, NU 7441(Tocris), an inhibitor of DNA-PK which blocks non-homologous end joining (NHEJ), was added to cells for 24 hours. Knock-in efficiency was evaluated using restriction fragment length polymorphism (RFLP) two days after transfection.
Delivery of Cas9 protein and gRNA into different cells
Cas9 protein and gRNA (2-part gRNA or sgRNA) were delivered into HeLa, RAW264.7, Jurkat, THP-1, K-562, and Raji cells using GenomONE-GE EX. Genome editing efficiency was evaluated using T7 endonuclease I mismatch cleavage assay two days after transfection.
Improvement of genome editing efficiency using chemically modified sgRNA
Unmodified or 2’OMe+PS-modified sgRNA was delivered with Cas9 protein into U-937 cells, respectively. Two days after transfection, genome editing efficiency was evaluated using T7 endonuclease I mismatch cleavage assay.
Improvement of cytotoxicity using detached floating cells in the transfection
HEK-293 cells were trypsinized, neutralized, centrifuged, and re-suspended in 10% fresh FBS culture medium. The detached floating cells were transfected with Cas9 protein and PPIB-targeting sgRNA using HVJ-E (GenomONE-GE EX ); centrifuged, re-resupended in 10% fresh FBS culture medium, and then transferred to 48-well culture plate. Genome editing efficiency was evaluated using T7 endonuclease I mismatch cleavage assay two days after transfection.
If severe cytotoxicity and/or many fused cells are observed in transfection of adherent cells such as HEK-293 cells, it is possible to reduce cytotoxicity and/or cell-to-cell fusion using detached floating cells in the transfection as described above.
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